The present invention relates in one aspect to bacterial strains and their preparations, the strains being capable of forming an alkaline phosphatase of the same species or also of a foreign species in greatly increased yields (as compared to the corresponding wild type). They are thus especially well suited for the fermentative production of these enzymes.
Processes for the isolation of alkaline phosphatases from bacteria are known from the monograph "The Enzymes", Ed. P.D. Boyer, Acad. Press (1971) IV: 373-415.
A plasmid is known having an E. Coli phosphatase gene which has an F' factor involved. It is produced by in vivo recombination of a fertility factor with a section of chromosomal DNA in E. Coli [M. Bracha, E. Yagil, J. Bacteriol. 120: 970-973 (1974)]. The isolation of such a plasmid has been described heretofore merely for the E. Coli phosphatase gene and does not represent a generally applicable method for the cloning of phosphatase genes. In particular, it is impossible in this way to clone genes of a quite different origin, e.g., from lower eucaryotes. The F'13 plasmid is present, as many other fertility factors, only in 1-2 copies per cell, whereby, also, only a minor stimulation of phosphatase formation is effected.
Alkaline phosphatases (EC3.1.3.1) are enzymes produced by a plurality of procaryotic and eucaryotic organisms. They are capable of hydrolyzing a great variety of phosphoric acid monoesters to the inorganic phosphate and the corresponding alcohol [Summary by T. W. Reid and I. B. Wilson in "The Enzymes" Ed. P.D. Boyer, Acad. Press (1971) IV: 373-415]. They are used in biochemical and molecular-biological research as well as in clinical diagnostics as preparative and analytical reagents.
DOS No. 2,617,350, for example, discloses a carrier-fixed alkaline phosphatase used in the production of defined oligonucleotides. A quantitative determination of phosphoric acid monoesters is possible by measuring the inorganic phosphate formed in the hydrolysis with alkaline phosphatase [Acta Chem. Scand. B31: 125-129 (1977)]. Another important application is the use of alkaline phosphatase as a readily detectable labeling enzyme in various enzyme immunoassays wherein it is coupled, depending on the type of analysis, to an antigen as well as to antibodies. Enzyme immunoassays permit a specific and quantitative determination of numerous substances (hormones, viral proteins, various cell wall antigens, antibodies, etc.) in minimal amounts [E. Engvall, A. J. Pesce, ed., "Quantitative Enzyme Immunoassay" Blackwell Scientific Publications, Oxford (1978)].
Alkaline phosphatase from E. coli is preferably utilized for many of these applications, wherein, inter alia, the high temperature stability of this enzyme (20 minutes at 85.degree. C.) as compared with mammalian phosphatases is of advantage (Japanese Patent J5 No. 2099-211).
Some bacteria, especially E. coli, synthesize individual enzymes in greatly increased amounts, if numerous copies of the corresponding gene per cell are present. Normally, the gene occurs only once per cell in wild-type bacteria [I. G. Young et al, "Gene" 4: 25-36 (1978)]. This high gene copy number (about 10-20 copies per cell) is attained by selectively transferring DNA fragments carrying the respective gene to bacteria with the aid of the techniques for in vitro recombination of DNA.
Various methods for the controlled transfer of genes to bacteria, also called DNA cloning, have been described in K. N. Timmis, S. N. Cohen, F. C. Caballo, "Progress in Molecular and Subcellular Biology", F. E. Hahn ed., 6: 1-58, Springer publishers (1978) whose disclosure is incorporated by reference herein. They are all based on the procedure of linking a DNA fragment carrying the respective gene to a vector DNA. It is then introduced in this form into a suitable receptor cell and replicated therein as recombinant DNA. Plasmids or bacteriophages serve as vectors. Plasmids are ring-shaped, double-strand DNA molecules found in some strains of bacteria. They are capable of replication therein independently of the chromosomal DNA (extrachromosomally). Essential aids in the cloning of DNA are the restriction endonucleases capable of sectioning double-strand DNA molecules into smaller fragments at exactly defined sites.
A frequently utilized process for the cloning of DNA fragments carrying genes for metabolic enzymes comprises, for example, reacting chromosomal DNA of a suitable donor organism and plasmid DNA with a suitable restriction endonuclease. The resultant fragments are then intermixed with DNA ligase, thus forming a plurality of different linear and circular, oligomeric products from the DNA fragments. Inter alia, these include ring molecules containing, per plasmid molecule, one molecule of the DNA fragment with the sought-after gene. The DNA mixture is then transferred in a process called transformation to suitable receptor bacteria. From the entire cell population, there are selected cells which contain the sought-after, recombined plasmid DNA. Variations of this procedure involve, for example, fragmenting of the chromosomal DNA by shear forces (ultrasound, etc.) and/or coupling of the DNA fragment to the plasmid via homopolymeric DNA single-strand termini produced with the enzyme terminal transferase [L. Clarke and J. Carbon, "Proc. Nat. Acad. Sci." (USA) 72: 4361-4365 (1975)]. Phage DNA has also been used successfully for cloning instead of plasmids.
In the technique of recombining DNA, especially when starting with a complex mixture of chromosomal DNA fragments, a large number of various hybrid molecules is usually produced in vitro. Therefore, a method of selecting a particular hybrid is required to enable cloning of defined DNA fragments, making it possible, after transformation and/or transfer, to isolate cell clones with the sought-after recombined DNA. If the fragment to be cloned carries a metabolic gene expressible in the receptor organism, then well-transformable mutants can be used as the recipients. These mutants contain the respective gene in inactivated form. Thus, they will exhibit the corresponding metabolic feature (i.e., will express the gene) only after they have absorbed the gene by transformation with recombined DNA in a replicatable form and thus in a form transmittable hereditarily. Accordingly, only those transformed mutant clones which in fact do express the desired gene under conventional translation conditions will have been transformed with the desired recombinant plasmid. The phosphatase-negative mutants of E. coli described in J. Bacteriol. 119: 583-592 (1974), in contradistinction to these required for the above purposes, involve strains which are poorly transformable due to their maintained capability for in vivo recombination. They are thus unsuitable for the cloning of phosphatase genes.